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bioRxiv · 10.1101/2025.04.25.650616

Mesoscopic SCAPE Microscope with a Rescanned, Super-oblique Illumination Plane

Cao, Z.·Jiapeng, Z.·Zhang, C.·Wang, Q.·Huang, Y.·Liu, W.·Shen, B.·Chai, Y.·Zhong, Z.·Li, H.·Wen, Q.·Wang, H.·Liang, W.
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Abstract

Investigating structural and functional dynamics across large spatial scales in living organisms calls for volumetric microscopy that combines a wide field of view (FOV), high spatial resolution, and sufficient temporal resolution. Swept confocally-aligned planar excitation (SCAPE) microscopy offers a compelling balance of optical sectioning, cellular resolution, and high-speed volumetric imaging. However, extending SCAPE to mesoscopic scales has been hindered by the limited tilting angle of the oblique illumination light sheet achievable with commercial low-magnification objectives. Here, we present a mesoscopic SCAPE microscope that overcomes this constraint by implementing a super-oblique light sheet--tilted up to ~60{degrees} relative to the optical axis of a 0.5-NA primary objective--enabled by an air-to-water bridging reflector. In addition, we introduce a single-galvanometer, self-conjugating rescanning strategy that allows arbitrary customization of the optical-to-mechanical angle ratio. This innovation accommodates the reversed and modulated scan displacement of the reflected light sheet and preserves descanned epi-fluorescence detection. Together, these advancements enable our mesoscopic SCAPE microscope to achieve a lateral FOV of 5.0 mm x 2.9 mm, cellular-level spatial resolution (~9.4 m x ~5.9 m lateral, and ~6.0 m axial), and a volumetric imaging rate of 9 volumes per second.

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Cao, Z., Jiapeng, Z., Zhang, C., Wang, Q., Huang, Y., Liu, W., Shen, B., Chai, Y., Zhong, Z., Li, H., Wen, Q., Wang, H., Liang, W.. 2025-04-28. Mesoscopic SCAPE Microscope with a Rescanned, Super-oblique Illumination Plane. https://doi.org/10.1101/2025.04.25.650616

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